Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 69 No. 3 pp. 545ñ549, 2012 ISSN 0001-6837Polish Pharmaceutical Society

Tephorsia purpurea Linn. (Fabaceae), com-monly known as Saraphunkha, is an importantindigenous medicinal plant used for centuries inIndian System of Medicine (ISM). It is an importantingredient of several marketed herbal formulationslike Tephroli and Yakrifit used for liver disorders.Various plant parts have been used as remedy forimpotency, asthma, diarrhoea, gonorrhoea, rheuma-tism, and urinary disorders (1). Experimental studiesshowed that T. purpurea also possessed antiulcer (2),antitumor (3), antilipid peroxidation activity (4).The plant has also been considered useful in biliousfibrile attacks as well as in obstruction of liver andspleen (5ñ8). The seed extract is reported to produceantihyperglycemic and antilipid peroxidation activi-ty in streptozotocin-induced diabetic rats (9). Thehepatoprotective potential of aerial plant parts hasbeen reported (10), in CCl4-induced hepatotoxicityanimal experimental model. Due to over exploita-tion of plant for the preparation of several herbalformulations, the plant is likely to become instinct.Hence, it was thought worthwhile to develop in vitrocultures of T. purpurea as an alternative source forthe production of plant material. In present investi-gation in vitro callus cultures of root of T. purpureawere developed on MS basal medium and its aque-ous extract was screened for antihepatotoxic poten-tial using CCl4 as toxicant. The antihepatotoxicpotential of in vitro produced callus cultures wasalso compared with that of natural root.

EXPERIMENTAL

Plant material

The fresh roots of Tephrosia purpurea Linnwere collected from the plant grown in HerbalGarden of Hamdard University in the month ofDecember and authenticated by taxonomist ofDepartment of Botany, Hamdard University.Voucher specimen was deposited in the Herbariumof plant tissue culture laboratories, Department ofPharmacognosy and Phytochemistry, HamdardUniversity, New Delhi.

Initiation and development of root callus ñThe aseptically germinated seedlings (11), wereinoculated on Murashige and Skoogís (MS) medi-um (12), supplemented with different concentra-tions and combinations of growth hormones and thegrowth was observed for 21 days. The hormonalcombination showing best callus initiation wasselected and the effect of different concentrationwas observed for development of calli. The rootcalli were developed and maintained for fivemonths The cultures were kept in B.O.D incubatorat 25 ± 2OC under 16-h diffused light ( 1600 lux ) /8-h darkness cycle .

Preparation of extracts

The natural root and 5 months old root calluswere dried below 60OC in an oven and powdered.The dried powdered root and root callus (10 g each)

SHORT COMMUNICATION

ANTIHEPATOTOXIC ACTIVITY OF AQUEOUS EXTRACTS OF CALLUSCULTURE OF TEPHROSIA PURPUREA (L) PERS

MOHD. MUJEEB1, RASHEEDUZ ZAFAR1, ASIF HUSAIN2* and AFTAB AHMAD3

1Plant Tissue Culture Laboratory, Department of Pharmacognosy and Phytochemistry, 2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard University,

New Delhi ñ 110 062, India3Jeddah Community College, King Abdul Aziz University, Jeddah, Kingdom of Saudi Arabia

Keywords: Tephrosia purpurea L. Pers, carbon tetrachloride, antihepatotoxic, root callus

545

* Corresponding author: e-mail: drasifhusain@yahoo.com, ahusain@jamiahamdard.ac.in

546 MOHD. MUJEEB et al.

were extracted separately with distilled water (100ml) for 4 h on water bath. The extracts obtainedwere filtered and evaporated to dryness underreduced pressure in rotary evaporator. The residuewas suspended in 1% of Tween 80 solution and usedfor the study.

Qualitative chemical analysis

The extracts of root and root callus were pre-pared and tested for the presence and absence of dif-ferent plant constituents by using standard qualita-tive chemical tests as follows: For alkaloids, 2 mLextract + 1% HCl + few drops of Mayerísreagents/Wagnerís reagent/Dragendroff reagentgave creamish white precipitate/brownish-red pre-cipitate/orange precipitate, respectively, which indi-cated the presence of alkaloids. For proteins, 2 mLextract + few drops of Millonís reagent gave a whiteprecipitate which turned red on heating, indicatingthe presence of proteins. For amino acids, 2 mLextract + few drops of ninhydrin reagent gave pink-ish red to violet color upon heating on water bath,indicating the presence of amino acids. For reducingsugars, 2 mL extract + 2 mL each of Fehling solu-tion A and B gave brick red precipitate upon heatingon water bath, indicating the presence of reducingsugars. For phenol, 2 mL extract + 2 mL FeCl3

(0.1% aqueous solution) gave blue-black precipitateindicating the presence of phenol. For saponins(frothing test), 0.5 mL filtrate + 5 mL distilled watergave frothing which persisted for some time, indi-cating the presence of saponins. For steroids(Liebermann-Burchard reaction), 2 mL extract + 2mL acetic anhydride + conc. H2SO4 gave blue-greenring, indicating the presence of steroids. Forflavonoids, 2 mL extract + conc. HCl + magnesiumribbon gave pink-tomato red color, indicating thepresence of flavonoids (11).

Chemicals

All chemicals used for the experiment were ofanalytical grade.

Animals

Non fasted Wistar strains of albino rats(140ñ200 g) of either sex supplied by animal houseof Hamdard University, New Delhi (RegistrationNo. 173/CPCSEA) were used. The animals wereallowed water and laboratory food ad libitum(Amrut laboratory rat and mice feed,Navamaharashtra Chakal oil mills Ltd., Pune). Thestudy was approved by ethics committee CPCSEA(Project no 68/2002). The animals were kept at 24OCand 40ñ70 RH with 12 h light period.

Anti-hepatotoxic activity

The animals were divided into five groups: Thefirst group (I) consisted of normal control rats whichreceived single daily dose of 1 mL/kg (i.p.) of 1%Tween 80 solution on all five days and olive oil 1mL/kg (s.c.) on days 2 and 3. The carbon tetrachlo-ride group (II) received single daily dose of 1 mL/kg(i.p.) of 1% Tween 80 solution (i.p.) for five daysand single dose of carbon tetrachloride 2 mL/kg(s.c.) on day 2 and 3. The third group (III) was treat-ed with 300 mg/kg (i.p.) of natural root extract on all5 days and carbon tetrachloride solution 2 mL/kg(s.c.) on day 2 and 3, 30 min after administration ofextract. The fourth group (IV) was treated with 300mg/kg (i.p.) of root callus extract on all 5 days andcarbon tetrachoride solution 2 mL/kg (s.c.) on day 2and 3, 30 min after administration of extract. Thefifth group (V) was treated with 10 mg/kg (i.p.) stan-dard (silymarin) on all 5 days and carbon tetrachlo-ride solution 2 ml/kg (s.c.) on days 2 and 3, 30 minafter administration of standard drug.

On final day, blood samples were withdrawnby heart puncturing. The blood samples wereallowed to clot for 30ñ40 min. Serum was separatedby centrifuging at 37OC ((g value: 1107)) and wasused for various biochemical estimations.

Estimation of serum alanine transaminase(ALT) and aspartate transaminase (AST) were basedon the reference method described by InternationalFederation of Clinical Chemistry. The reagents sup-plied in the kits (Span Diagnostics Kits) were recon-stituted, mixed with serum as directed. The ALT andAST were measured at 340 nm and expressed asIU/mL (13). The serum alkaline phosphatase (ALP)was estimated by mixing with the reagent (p-nitro-phenyl phosphate, magnesium, buffers and stabiliz-ers) with serum, estimated at 405 nm and expressedas IU/mL (14). Total bilirubin was estimated byMalloy and Evelyn method (15) measured at 546 nmand expressed as mg/dL. Biuret method (16) wasfollowed for the estimation of total protein. Serumwas mixed with biuret reagent and incubated for 10min at 37OC. The total protein was estimated at 555nm and expressed as g/dL.

Histological studies

Livers were quickly removed and preserved inneutral buffered formalin. Histological liver sectionswere prepared as described previously by Luna (17).

Statistical analysis

Values are expressed as the mean ± SEM.Statistical analysis was performed by one-wayanalysis of variance (ANOVA), followed by

Antihepatotoxic activity of aqueous extracts of callus culture of... 547

Dunnett test. The values were considered statistical-ly significant, if p was less than 0.05.

RESULTS AND DISCUSSION

The root callus of Tephrosia purpurea was suc-cessfully developed and maintained on MS mediumsupplemented with 2,4-dichlorophenoxy acetic acid(4.52 µM), indole acetic acid (28.5 µM) and kinetin(9.29 µM) for five months. The callus produced wassoft in texture and creamy white in color.Preliminary phytochemical screening of aqueousextracts of root and root callus reveals the presenceof alkaloids, proteins, amino acids, phenolic com-pounds, reducing sugars, steroids, flavonoids, andsaponin glycosides.

In the present study, treatment of rats with rootcallus extract at a dose of 300 mg/kg b.w., i.p., pro-duced highly significant (p < 0.001) reduction inelevated serum enzymes level (AST, ALT and ALP)and bilirubin content, moreover, it also showed abetter increase in total protein level as compared totoxic control group II (Tab. 1). Root extract at a doseof 300 mg/kg b.w. i.p. showed lower anti-hepato-toxic activity. The anti-hepatotoxic activity of rootcallus extract was compared with natural rootextract (300 mg/kg) and standard drug silymarinextract (10 mg/kg). It was found that the root callusextracts showed better protection against CCl4-induced liver damage as compared to natural rootextract. The anti-hepatotoxic activity of the root cal-lus extract was found to be comparable to that ofstandard drug (silymarin).

T. purpurea Linn. is used in the treatment ofvarious liver diseases including hepatitis and jaun-dice in Ayurveda and Unani System of Medicine(7). In the present investigation, the anti-hepatotox-ic activity of aqueous extracts of root and root calluswere evaluated in rats by using CCl4-induced hepa-totoxicity model. The hepatic damage caused byCCl4 is evident from the elevated level of the hepat-ic enzymes (ALT, AST and ALP) and bilirubin con-tent in CCl4-treated group as compared to controlgroup, where serum total protein levels were con-siderably reduced by carbon tetrachloride treatment(Tab. 1). These biochemical parameters are usefulfor the detection of early damage of hepatic tissuesand require fewer efforts than those required for his-tologic analyses, moreover, without sacrifice of theanimals. The elevated level of serum transaminases(ALT, AST and ALP) reflects the damage to thestructural integrity of liver (18), as tranaminases arefound in cytoplasm and released into circulationafter cellular damage (19). The biotransformation of

Tab

le 1

.E

ffec

t of

aque

ous

extr

acts

of

root

and

roo

t cal

lus

of T

ephr

osia

pur

pure

aon

var

ious

bio

chem

ical

par

amet

ers.

Gro

ups

Tre

atm

ent

Dos

e an

d ro

ot

AST

(U

/mL

) A

LT

(U

/mL

) A

LP

(IU

/mL

) T

B (

mg/

100

mL

) T

P (g

/100

mL

)

IC

ontr

ol1

mL

/kg

(i.p

.)50

.00

± 1.

0560

.66

± 1.

0334

.66

± 0.

970.

381

± 0.

004

8.70

± 0

.25

IIT

oxic

con

trol

2 m

L/k

g (s

.c.)

680.

23 ±

15.

1134

0.15

± 8

.33

280.

23 ±

6.0

87.

981

± 2.

445.

85 ±

0.5

1

III

Roo

t ext

ract

300

mg/

kg (

i.p.)

290.

15 ±

7.0

8 *

248.

33 ±

6.1

2 *

150.

45 ±

4.0

5*1.

723

± 0.

07 *

*6.

73 ±

0.8

5 *

IVR

oot c

allu

s ex

trac

t30

0 m

g/kg

(i.p

.)10

0.01

± 4

.16

***

90.2

2 ±

2.25

**

80.3

2 ±

1.23

**0.

212

± 0.

08 *

**8.

16 ±

0.4

5 **

*

VSi

lym

arin

10 m

g/kg

(i.p

.)56

.11

± 1.

65 *

**60

.22

± 1.

45 *

*70

.04

± 2.

33**

0.35

1 ±

0.02

***

9.34

± 0

.36

***

The

val

ues

repr

esen

t the

mea

n ±

SE. n

= 6

* p

< 0

.05,

**

p <

0.01

, ***

p <

0.0

01 v

s.to

xic

cont

rol (

Gro

up I

I) (

AN

OV

A f

ollo

wed

by

Dun

nett

test

) A

LT

: Ala

nine

tran

sam

inas

e, A

ST: A

spar

tate

tran

sam

inas

e, A

LP:

Alk

alin

e ph

osph

atas

e, T

B: T

otal

bili

rubi

n, T

P: T

otal

pro

tein

s.

548 MOHD. MUJEEB et al.

carbon tetrachloride leads to the formation of theactive free radical CCl3 and this free radical reactsrapidly with oxygen to form a trichloromethylper-oxy radical, which may contribute to the hepatotox-icity and subsequent increase in hepatic enzymes(20). This is further supported by the elevation in the

level of AST, ALT and ALP in carbon tetrachloridetreated rats. The natural root and root callus in adose of 300 mg/kg protect the animals significantlyfrom CCl4-induced hepatotoxicity as compared totoxic control group. This was evident by the signifi-cant reduction in serum ALT, AST, ALP and biliru-bin content. There was no significant differencebetween the root callus extract group and standarddrug group. The above findings can be correlatedwith those of an earlier study (21) in which T. pur-purea extract exerted hepatoprotective actionagainst CCl4 hepatotoxic model. The anti-hepatotox-ic activity of the natural root and root callus extractcould be due to the presence of flavonoids glyco-sides which have hepatoprotective and anti-oxidantproperties (22, 23). In the present study, preliminaryphytochemical screening of T. purpurea revealed thepresence of flavonoid glycosides.

Histological observations

Histopathological studies of liver sections incontrol animals showed normal hepatic cells with

Figure 1. Liver tissue of control animal showing normal histology Figure 2. Liver tissue of CCl4 treated animal showing necrosis,necrotic cells and fatty changes

Figure 3. Liver tissue of animal treated with CCl4 and T. purpurea(300 mg/kg) of root showing moderate necrosis, mild fattychanges

Figure 5. Liver tissue of rats treated with CCl4 and silymarin (10mg/kg) showing almost normal histology

Figure 4. Liver tissue of animal treated with CCl4 and T. purpurea(200 mg/kg) of root callus showing very mild fatty changes

Antihepatotoxic activity of aqueous extracts of callus culture of... 549

prominent nucleus and central vein (Fig. 1). In CCl4-treated animals the sections showed hydropicchanges in centrilobular hepatocytes with single cellnecrosis, congestion of central vein and sinusoidswere seen with acute and chronic inflammatory cellsmainly in central zone (Fig. 2). Treatment of theanimals with silymarin and root and root callusextract of T. purpurea extract exhibited a significantrecovery of hepatocytes in different sections of theliver (Figs. 3ñ5).

Histopathological studies revealed that CCl4

caused steatosis and hydropic degeneration of theliver tissue. Root and root callus extract of T. pur-purea treatment exhibited protection, which con-firmed the result of biochemical studies. Also all theeffects of root and root callus extract of T. purpureawere comparable with those of silymarin, a provenhepatoprotective drug.

CONCLUSION

On the basis of above results, it can be con-cluded that the active principles present in root cal-lus extract of T. purpurea offered better antihepato-toxic action as compared to the active principlespresent in natural root extract against CCl4 hepaticdamage

However, more elaborate work is required toestablish the efficacy of root callus extract by isolat-ing and identifying the active constituents present inthe natural root and root callus extracts which areresponsible for antihepatotoxic activity.

Acknowledgments

We are thankful to University GrantCommission (UGC) and Jamia Hamdard for finan-cial assistance. Thanks are also due to Mr. QaisarNaeem, Department of Reprography, JamiaHamdard, New Delhi for his help.

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Received: 2. 10. 2010